A classical example of the problems in this field is encountered during the determination of retroviral reverse transcriptase (RT) in cell extracts or cell culture fluids. During reverse transcription the RT produces a DNA copy of the viral RNA. A conventional RT activity assay is performed by utilizing an artificial template-primer construction and tritiated deoxynucleotide triphosphate as nucleotide substrate. The template/primer pair poly(rA)/oligo(dT) is the most efficient and most used combination for determination of HIV as well as for other retroviral RTs (Baltimore-71, Lee et al-87). It was early recognized that some cellular DNA polymerizing enzymes obscured the results of this type of assay system by producing measurable product amounts.
At least nine different types of DNA polymerases have been identified in eukaryotic cells. Polymerase α is considered to be responsible for DNA replication during cell division, β is regarded as being the major polymerase involved in repair of DNA and λ is responsible for DNA synthesis in mitochondria. Each of the cellular polymerases occurs in different forms and with different associated proteins. Their enzymatic properties vary both with their molecular form and with cell type origin (for a review see Hübscher et al 2000). In addition, cells may contain enzymes derived from infecting viruses or endogenous virus genes that encode viral DNA polymerase. Of the cellular DNA polymerases, DNA polymerase α is least prone to utilize prA as template, but there are certain molecular species of this enzyme that can efficiently copy prA (Goulian & Grimm 1990, Yoshida et al 1981).
In effect the different polymerases represent a specificity problem during quantification of retroviral RT. Many methods to circumvent this problem can be found in the literature. The most obvious approach is to separate the virus from the cellular proteins. Various methods such as centrifugation or adsorption of virions to specific receptors or antibodies have been used in this context. A common problem occurring when purifying material containing large amounts of cellular components and minute quantities of virus is that co-purification of small amounts of cellular polymerase cannot be excluded. Furthermore, from a practical point of view it is not attractive to process large amounts of samples with relatively cumbersome separation procedures.
Another approach is to design isozyme specific enzyme assay conditions which more or less exclude cellular polymerase, i.e. λ polymerase, from detection. This can be achieved by using inhibitors, alternative metal ions or template/primer pairs. For example poly(2′-0-methyl-rC)n oligo(dG)12-18 is known to be a highly specific substrate for retroviral RTs. The increased specificity achieved by this type of method is, however, usually hampered by the corresponding decrease in reaction velocity and subsequent reduction in detection sensitivity for the enzyme that is intended to be measured.
We have recently developed a method for quantification of HIV RT in plasma sampled from infected persons. The method is based on binding of the virus to a gel with an immobilized ion exchanger. Antibodies and disturbing substances are removed by a wash and enzymatically active RT is recovered by lysis of the immobilized virus with non-ionic detergent (Gatu et al 2000). The amount of RT recovered is finally quantified with a sensitive RT assay based on the use of immobilized prA primed with odT. When processing control plasma from healthy blood donors in this system we found small amounts of RT activity in the purified fractions.
Further investigations revealed that crude EDTA plasma from healthy humans contained comparatively large amounts of this activity, which also was abundant in extracts from the lymphocyte fraction of the same type of samples. Some characteristics of this unclassified enzyme are depicted in Table 1. The amount of activity recovered from our separation system after “virus immobilization” was less than a thousandth part of the total activity in plasma but enough to severely obscure the detection of small amounts of HIV RT.